Summary: Our laboratory studies signal transduction in the immune system with a focus on tyrosine non-receptor tyrosine kinases and downstream effector molecules. These proteins are required for intracellular signaling pathways involved in normal cellular growth and differentiation as well as the abnormal growth, differentiation and development involved in to multiple human disorders including primary immunodeficiencies, autoimmunity and cancer. Using a combination of genetics, protein biochemistry and cell biology, our goals are to understand how signaling molecules and pathways contribute to normal function of cells in the immune system and their dysfunction in disease, and how manipulation of signaling pathways can aid in the development of therapeutics. We are particularly interested in how these pathways affect responses to infectious diseases and immunization. Research: I. Tec Kinases: Over the last 12 years, we have studied members of the Tec family of tyrosine kinases. Mutations affecting the prototypical member, Btk, are responsible for the human genetic disorder X-linked agammmaglobulimemia, charccterized by abnormal B cell development and function. We have shown that mutations affecting Tec kinases expressed in T cells, Itk and Rlk, impair T lymphocyte development and function in mice and alter responses to infections in vivo. In addition to their recognized roles in T cell receptor induced activation of phospholipase-c gamma and Ca++ mobilization, we have shown that the Tec kinases play important roles in regulating the actin cytoskeleton and cell adhesion, which are critical for T cells to exert effector cell functions. We have also shown that mutations of these kinases affect patterns of cytokine production by CD4+ T helper lymphocytes and the ability of mice to respond to distinct types of infectious diseases. Confirming the importance of these kinases, another group reported this year that two siblings with a profound lethal immunodeficiency had mutations affecting Itk. Over the last year, we have further examined the role of Tec kinases in CD4+ T helper cell differentiation and regulation of patterns of cytokine production important for distinct types of immune responses. We and others have previously demonstrated that Itk, the Tec kinase most highly expressed in T cells, is required for TH2 cytokine production and in vivo TH2 responses, a subclass of responses important for certain parasitic infections as well as allergens. In contrast, expression of Rlk/Txk has been linked to TH1 responses (important for inflammation and responses to intracellular pathogens). In work published in this reporting period, we characterized the role of Tec kinases in regulating expression of IL-17 and differentiation of TH17 cells, an effector cell subclass important for responses to extracellular bacteria and which contribute to the pathology of autoimmune and inflammatory disorders. We find that TCR signaling plays a critical role in the differential regulation of IL-17A and IL-17F, two related cytokines expressed by TH17 cells, via a pathway involving Itk-mediated activation of the Ca2+ sensitive transcription factor NFATc1. Thus, although the genes encoding both cytokines are linked and undergo similar epigenetic modifications, in the absence of strong TCR signaling or Itk, IL-17A is less-well transcribed due to decreased NFATc1 DNA binding. Although IL-17A and IL-17F are often thought to be similarly regulated, IL-17A is much more inflammatory and can be expressed by a more restriicted pattern of TH17 cells. Our work provides evidence that TCR signaling provides a second level of regulation of this very pro-inflammatory and potentially tissue-damaging cytokine (Gomez-Rodriguez et al. Immunity, 2009). II. SAP: As an extension of these studies, we are examining other signaling molecules involved in T helper cell differentiation including SAP, a small SH2 containing adaptor protein, mutations of which are associated with the genetic disorder X-linked proliferative syndrome (XLP). SAP binds to and helps recruit the tyrosine kinase Fyn to the intracellular tails of SLAM and related co-stimulatory receptors. We previously generated mice deficient in SAP and found that upon challenge with infectious agents, these mice recapitulated features of XLP, including increased T cell activation and decreased antibody production. SAP is required for TH2 cytokine (IL-4) expression in response to TCR stimulation. We have shown this function is abrogated by mutations affecting the region required to recruit Fyn. In this reporting period, we have extended our findings to show that SAP also interacts with PKC-theta and that PKC-theta is required for SLAM and SAP-mediated regulation of IL-4 expression (Cannons et al, J. Immunol. 2010). In the last several years, we have demonstrated that SAP also plays a critical role in responses to immunization. We further showed that the impaired antibody responses are T cell intrinsic, ie SAP deficient T cells fail to provide essential signals to B cells for generating germinal centers and long-term antibody responses, which are critical for successful vaccination. In collaboration with Dr. R. Germain of NIAID, we used intravital microscopy to show that SAP-deficient CD4+ T cells have a selective defect in adhesion to B cells in vivo and in vitro, preventing them from delivering the contact-dependent signals required for B cells to form germinal centers and generate long-term humoral responses. This work, (Qi et al, Nature, 2008), provided new insight into the requirements for T:B cell interactions and cellular collaborations required for long-term antibody responses, a critical feature of successful immunization. Moreover, it provided potential insight into the B-cell centric phenotypes in XLP, including defective T cell help for B cell antibody responses and an inability of T cells and NK cells to kill EBV-infected B cells (Schwartzberg, Nat Rev. Immunol 2009). In unpublished work, we have confirmed these adhesion defects in cells from XLP patients. We have continued in this work and have shown that T;B cell interactions have both an early integrin-dependent phase and a later phase that was dependent on SAP. Moreover, the ability of SAP to mediate adhesion is independent of its ability to recruit Fyn (in contrast to cytokine regulation), but dependent on the ability of SAP to bind SLAM family members through its SH2 domain. Thus, SAP-mediated adhesion may involve distinct signaling pathways, perhaps downstream of distinct SLAM family members. We generated CD84-deficient mice, revealing that CD84 is a key SLAM family member involved in T:B cell interactions required for germinal center formation that functions as an adhesion molecule required for T:B cell, but not T:DC conjugation. However, the defect in CD84-deficient mice is partial--our data further suggests that the SLAM family member Ly108 is another adhesive receptor for T:B cell interactions, linking multiple family members to this phenotype and providing windows for therapeutic development (Cannons et al, Immunity 2010). We are continuing this work, focusing on the role of SAP in Tfh cells required for germinal center formation. Although our studies are primarily performed in mice, our results have provided the basis for several confirmatory studies showing similar defects in germinal centers, long term antibody and B cell memory responses in XLP patients. Understanding the cellular interactions and signals defective in these mice is therefore of high importance for understanding the pathophysiology of this disease as well as the requirements for successful vaccine development.